A current issue in the clinical management of prostate cancer, the second most deadly malignancy in American men, is the need to accurately distinguish between indolent and aggressive forms of the disease to inform the treatment course. Very little is known about the molecular differences between these two forms of the disease, and this knowledge is vital for the development of improved treatments and diagnostics. DNA methylation is a chemical modification that occurs in the human genome, and has been found to differ greatly between benign tissue and tumor tissue. DNA methylation is well studied in one context, called promoter CpG islands, where it can suppress the expression, or activity, of genes. However, preliminary data suggests that much of the DNA methylation differences between indolent and aggressive prostate cancer cases are not in these locations, and very little is known about DNA methylation in these other locations and how it can affect genes and lead to different levels of disease aggressiveness. The central hypothesis of this work is that DNA methylation can drive the indolent and aggressive phenotypes in prostate cancer by altering the binding sites of regulatory proteins, and that some of these sites change the activity of distant genes. The study proposes to replicate and characterize methylation differences observed in a preliminary cohort in a larger cohort, and using a higher-resolution method, confirm that these methylation differences are unique to aggressive prostate cancer. This high-resolution data will be used to computationally infer which DNA-protein binding sites may be altered by the methylation differences. Next, this study will experimentally determine the functional mechanisms within gene body DNA methylation in conferring tumor aggressiveness by directly altering methylation at these sites. In human cultured cell lines, a genome editing technology will be employed that can target a protein that either removes or adds methylation to a specific site in the genome. It is proposed to use this editing technology to manipulate two regions that contain methylation only in high grade cases that were found in the preliminary cohort and that show evidence of replication in The Cancer Genome Atlas (TCGA). A sequencing technique will be used to find distant genes, that while distant in linear space, are nearby in three-dimensional space. Assays will be performed to test for changes in gene expression of these genes and for changes in aggressive cellular behavior. This will establish the functional activity of these regions and connect this change to gene expression and aggressiveness to augment our understanding of the specific mechanisms of how cancer aggressiveness is regulated by DNA methylation. The impact of this work is that the problem of the molecular basis of prostate cancer aggressiveness will be addressed while providing insight into the basic biology of DNA methylation and the specific pathways through which it can confer aggressiveness to tumors. These methylation changes can both serve as diagnostic tools and inform the development of new therapies.